Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells.

Cytotoxic CD8 +T lymphocytes (CTLs) are key players of adaptive anti-tumor immunity based on their ability to specifically recognize and destroy tumor cells. Many cancer immunotherapies rely on unleashing CTL function. However, tumors can evade killing through strategies which are not yet fully elucidated. To provide deeper insight into tumor evasion mechanisms in an antigen-dependent manner, we established a human co-culture system composed of tumor and primary immune cells. Using this system, we systematically investigated intrinsic regulators of tumor resistance by conducting a complementary CRISPR screen approach. By harnessing CRISPR activation (CRISPRa) and CRISPR knockout (KO) technology in parallel, we investigated gene gain-of-function as well as loss-of-function across genes with annotated function in a colon carcinoma cell line. CRISPRa and CRISPR KO screens uncovered 187 and 704 hits, respectively, with 60 gene hits overlapping between both. These data confirmed the role of interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α) and autophagy pathways and uncovered novel genes implicated in tumor resistance to killing. Notably, we discovered that ILKAP encoding the integrin-linked kinase-associated serine/threonine phosphatase 2 C, a gene previously unknown to play a role in antigen specific CTL-mediated killing, mediate tumor resistance independently from regulating antigen presentation, IFN-γ or TNF-α responsiveness. Moreover, our work describes the contrasting role of soluble and membrane-bound ICAM-1 in regulating tumor cell killing. The deficiency of membrane-bound ICAM-1 (mICAM-1) or the overexpression of soluble ICAM-1 (sICAM-1) induced resistance to CTL killing, whereas PD-L1 overexpression had no impact. These results highlight the essential role of ICAM-1 at the immunological synapse between tumor and CTL and the antagonist function of sICAM-1.

Reporting Standards for Diagnostic Testing: Guidance for Authors From Editors of Respiratory, Sleep, and Critical Care Journals.

Diagnostic testing is fundamental to medicine. However, studies of diagnostic testing in respiratory medicine vary significantly in terms of their methodology, definitions, and reporting of results. This has led to often conflicting or ambiguous results. To address this issue, a group of 20 respiratory journal editors worked to develop reporting standards for studies of diagnostic testing based on a rigorous methodology to guide authors, peer reviewers, and researchers when conducting studies of diagnostic testing in respiratory medicine. Four key areas are covered, including defining the reference standard of truth, measures of dichotomous test performance when used for dichotomous outcomes, measures of multichotomous test performance for dichotomous outcomes, and what constitutes a useful definition of diagnostic yield. The importance of using contingency tables for reporting results is addressed with examples from the literature. A practical checklist is provided as well for reporting studies of diagnostic testing.

Development of a Bispecific Antibody-Based Platform for Retargeting of Capsid Modified AAV Vectors.

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope "2E3" derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins-fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches.

Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells.

Bone morphogenetic protein 9 (BMP9) and BMP10 are circulating ligands that mediate endothelial cell (EC) protection via complexes of the type I receptor ALK1 and the type II receptors activin type-IIA receptor (ACTR-IIA) and bone morphogenetic type II receptor (BMPR-II). We previously demonstrated that BMP9 induces the expression of interleukin-6, interleukin-8 and E-selectin in ECs and might influence their interactions with monocytes and neutrophils. We asked whether BMP9 and BMP10 regulate the expression of chemokine (C-C motif) ligand 2 (CCL2), a key chemokine involved in monocyte-macrophage chemoattraction. Here, we show that BMP9 and BMP10 repress basal CCL2 expression and release from human pulmonary artery ECs and aortic ECs. The repression was dependent on ALK1 and co-dependent on ACTR-IIA and BMPR-II. Assessment of canonical Smad signalling indicated a reliance of this response on Smad4. Of note, Smad1/5 signalling contributed only at BMP9 concentrations similar to those in the circulation. In the context of inflammation, BMP9 did not alter the induction of CCL2 by TNF-α. As CCL2 promotes monocyte/macrophage chemotaxis and endothelial permeability, these data support the concept that BMP9 preserves basal endothelial integrity.

British Thoracic Society Training Standards for Thoracic Ultrasound (TUS).

INTRODUCTION: The British Thoracic Society (BTS) responded to a call from the pleural community to establish this new Training Standard to detail the capabilities in practice for thoracic ultrasound (TUS), which will build on the previous curricula and extend the remit to include training for the emergency provision of TUS. METHODS: BTS convened a working group to produce a set of Training Standards. RESULTS: This document provides a comprehensive Training Standard for TUS facilitating timely and improved management of patients with respiratory presentations, particularly (but not exclusively) pleural pathologies. DISCUSSION: The Training Standards document will be widely disseminated.

Pulmonary venous hypertension and mechanical strain stimulate monocyte chemoattractant protein-1 release and structural remodelling of the lung in human and rodent chronic heart failure models.

INTRODUCTION: The burden of chronic heart failure (HF) is rising owing to an increased survivorship after myocardial infarction (MI). Pulmonary structural remodelling in patients with HF may protect against oedema while causing dyspnoea, the predominant symptom associated with HF. The cellular and molecular mechanisms underlying these processes in HF are poorly understood. We hypothesised that pulmonary venous hypertension (PVH) following MI provides a mechanical stimulus for structural remodelling of the lung via monocyte chemoattractant protein-1 (MCP-1). METHODS: Human lung microvascular endothelial cells (HLMVEC) and Ea.Hy 926 cells exposed to cyclic mechanical strain (CMS) in vitro were analysed for MCP-1 expression and activation of signalling intermediates. HF was induced in Sprague-Dawley rats 16 weeks after MI; a cohort was rescued with AAV9.SERCA2a gene therapy to reduce PVH. RESULTS: HLMVEC and Ea.Hy 926 cells exposed to CMS upregulated MCP-1 gene expression and protein release in an extracellular-signal-regulated kinase (ERK) 1/2 dependent manner. Supernatants from these experiments stimulated fibroblast (human fetal lung fibroblast -1) and pulmonary artery smooth muscle cell proliferation and differentiation. Total lung collagen, a marker of structural remodelling, and MCP-1 gene expression were increased in the lungs of rats with post-MI HF. SERCA2a gene therapy that attenuated PVH after MI was associated with lower levels of lung collagen and MCP-1 gene expression in the lung. CONCLUSIONS: Mechanical strain associated with PVH may stimulate pulmonary structural remodelling through ERK 1/2 dependent induction of MCP-1. These findings provide insights into the pathophysiology of lung remodelling in HF and highlight novel, potential therapeutic targets.

Molecular structure of human GM-CSF in complex with a disease-associated anti-human GM-CSF autoantibody and its potential biological implications.

Polyclonal autoantibodies against human GM-CSF (granulocyte/macrophage colony-stimulating factor) are a hallmark of PAP (pulmonary alveolar proteinosis) and several other reported autoimmune diseases. MB007 is a high-affinity anti-(human GM-CSF) autoantibody isolated from a patient suffering from PAP which shows only modest neutralization of GM-CSF bioactivity. We describe the first crystal structure of a cytokine-directed human IgG1λ autoantibody-binding fragment (Fab) at 1.9 Å (1 Å=0.1 nm) resolution. Its CDR3-H substantially differs from all VH7 germline IgG1 structures reported previously. We derive a reliable model of the antigen-autoantibody complex by using NMR chemical shift perturbation data in combination with computational methods. Superposition of the modelled complex structure with the human GM-CSF-GM-CSF ternary receptor complex reveals only little overlap between receptor and Fab when bound to GM-CSF. Our model provides a structural basis for understanding the mode of action of the MB007 autoantibody.

BMP-9 induced endothelial cell tubule formation and inhibition of migration involves Smad1 driven endothelin-1 production.

BACKGROUND: Bone morphogenetic proteins (BMPs) and their receptors, such as bone morphogenetic protein receptor (BMPR) II, have been implicated in a wide variety of disorders including pulmonary arterial hypertension (PAH). Similarly, endothelin-1 (ET-1), a mitogen and vasoconstrictor, is upregulated in PAH and endothelin receptor antagonists are used in its treatment. We sought to determine whether there is crosstalk between BMP signalling and the ET-1 axis in human pulmonary artery endothelial cells (HPAECs), possible mechanisms involved in such crosstalk and functional consequences thereof. METHODOLOGY/PRINCIPAL FINDING: Using western blot, real time RT-PCR, ELISA and small RNA interference methods we provide evidence that in HPAECs BMP-9, but not BMP-2, -4 and -6 significantly stimulated ET-1 release under physiological concentrations. This release is mediated by both Smad1 and p38 MAPK and is independent of the canonical Smad4 pathway. Moreover, knocking down the ALK1 receptor or BMPR II attenuates BMP-9 stimulated ET-1 release, whilst causing a significant increase in prepro ET-1 mRNA transcription and mature peptide release. Finally, BMP-9 induced ET-1 release is involved in both inhibition of endothelial cell migration and promotion of tubule formation. CONCLUSIONS/SIGNIFICANCE: Although our data does not support an important role for BMP-9 as a source of increased endothelial ET-1 production seen in human PAH, BMP-9 stimulated ET-1 production is likely to be important in angiogenesis and vascular stability. However, increased ET-1 production by endothelial cells as a consequence of BMPR II dysfunction may be clinically relevant in the pathogenesis of PAH.

The role of endothelin-1 in the pathogenesis of pulmonary arterial hypertension.

The term pulmonary arterial hypertension (PAH) describes a rare group of diseases characterized by raised pulmonary vascular resistance, resulting from vascular remodelling in the pre-capillary resistance arterioles (< 100 mm). Left untreated, patients die from right heart failure, with a mortality approaching most serious cancers. Endothelin-1(ET-1) is not only a potent vasoconstrictor, but causes proliferation of many of the vascular cells involved in vascular remodelling. Although produced mainly by the vascular endothelium, other cells such as smooth muscle, fibroblasts and macrophages are known sources of ET-1 when these cells are challenged by relevant stimuli. Plasma ET-1 levels are raised in patients with PAH and correlate with important clinical outcomes. Furthermore, ET-1 receptor antagonism has been demonstrated to improve both morbidity and mortality in conditions associated with PAH. We review the literature supporting the role for ET-1 in the pathogenesis of PAH.

Phenocopy--a strategy to qualify chemical compounds during hit-to-lead and/or lead optimization.

A phenocopy is defined as an environmentally induced phenotype of one individual which is identical to the genotype-determined phenotype of another individual. The phenocopy phenomenon has been translated to the drug discovery process as phenotypes produced by the treatment of biological systems with new chemical entities (NCE) may resemble environmentally induced phenotypic modifications. Various new chemical entities exerting inhibition of the kinase activity of Transforming Growth Factor ß Receptor I (TGF-ßR1) were qualified by high-throughput RNA expression profiling. This chemical genomics approach resulted in a precise time-dependent insight to the TGF-ß biology and allowed furthermore a comprehensive analysis of each NCE's off-target effects. The evaluation of off-target effects by the phenocopy approach allows a more accurate and integrated view on optimized compounds, supplementing classical biological evaluation parameters such as potency and selectivity. It has therefore the potential to become a novel method for ranking compounds during various drug discovery phases.

Design, synthesis, and evaluation of indolinones as inhibitors of the transforming growth factor ß receptor I (TGFßRI).

Inhibition of transforming growth factor ß (TGFß) type I receptor (Alk5) offers a novel approach for the treatment of fibrotic diseases and cancer. Indolinones substituted in position 6 were identified as a new chemotype inhibiting TGFßRI concomitant with a low cross-reactivity among the human kinome. A subset of compounds showed additional inhibition of platelet-derived growth factor receptor alpha (PDGFRα), contributing to an interesting pharmacological profile. In contrast, p38 kinase, which is often inhibited by TGFßRI inhibitors, was not targeted by derivatives based on the indolinone chemotype. Guided by an X-ray structure of lead compound 5 (BIBF0775) soaked into the kinase domain of TGFßRI, optimization furnished potent and selective inhibitors of TGFßRI. Potent inhibition translated well into good inhibition of TGFßRI-mediated phosphorylation of Smad2/3, demonstrating efficacy in a cellular setting. Optimized compounds were extensively profiled on a 232-kinase panel and showed low cross-reactivities within the human kinome.

Reference gene selection for real-time polymerase chain reaction in human lung cells subjected to cyclic mechanical strain.

BACKGROUND AND OBJECTIVE: The respiratory system is constantly exposed to mechanical forces that influence cellular phenotype in health and disease. Quantitative real-time PCR (qPCR) is widely used to determine gene expression. The validity of qPCR depends on using stable reference genes for normalization. The effect of cyclic mechanical strain on reference gene expression by lung epithelial, fibroblast and endothelial cells has not been studied systematically. METHODS: The stability of expression of fourteen potential reference genes in response to six different regimens of cyclic mechanical strain was ranked using the geNorm tool in human lung epithelial cell lines (A549 and H441), human fetal lung fibroblasts (HFL-1), human lung microvascular endothelial cells, primary human lung fibroblasts and primary human alveolar type 2 (hAT2) cells. The expression variation of these reference genes was also screened in unstimulated whole human lung. RESULTS: The stability of the selected reference genes varied within and between cell types, the variation in expression being greatest in primary cultures of hAT2. Correspondingly, the effect of expressing message for the stretch responsive gene IL-8 normalized to the 14 reference genes was greatest in the hAT2 cells, there being an almost fivefold difference in mRNA relative change comparing different reference genes in the same samples. The minimum number of genes required to derive a reliable normalization factor for experiments on single lung cell types undergoing mechanical strain was two and for whole human lung it was four. CONCLUSIONS: These results demonstrate that the optimal reference genes for lung cells subjected to CMS are cell type specific.

Pharmacologic differentiation of inflammation and fibrosis in the rat bleomycin model.

RATIONALE: The model most often used to study the pathogenesis of pulmonary fibroses is the bleomycin (BLM)-induced lung fibrosis model. Several treatments have been efficacious in this model, but not in the clinic. OBJECTIVES: To describe the time course of inflammation and fibrosis in the BLM model and to study the effect of timing of antiinflammatory and antifibrotic treatments on efficacy. METHODS AND MEASUREMENTS: Rats were given single intratracheal injections of BLM on Day 0. At specified time points, 10 rats were killed and their lungs studied for proinflammatory cytokines and for profibrotic growth factor mRNA. After a single intratracheal injection of BLM on Day 0, rats were treated from Day 1 or 10 daily with oral prednisolone (10 mg/kg) or oral imatinib mesylate (50 mg/kg) for 21 d. RESULTS: After BLM administration, the expression of inflammatory cytokines was elevated and returned to background levels at later time points. Profibrotic gene expression peaked between Days 9 and 14 and remained elevated till the end of the experiment, suggesting a "switch" between inflammation and fibrosis in this interval. Antiinflammatory treatment (oral prednisolone) was beneficial when commenced at Day 1, but had no effect if administered from Day 10 onward. However, imatinib mesylate was effective independently of the dosing regime. CONCLUSIONS: The response of the BLM model to antifibrotic or antiinflammatory interventions is critically dependent on timing after the initial injury.

Early haemodynamic benefit of sildenafil in patients with coexisting chronic thromboembolic pulmonary hypertension and left ventricular dysfunction.

Sildenafil, a phosphodiesterase type-5 inhibitor, offers potential to treat pulmonary hypertension associated with a variety of conditions. We assessed the early impact of sildenafil on a cohort of patients referred to our unit who had severe pulmonary hypertension secondary to chronic thromboembolic disease which was not amenable to pulmonary thromboendarterectomy and who also had coexisting left ventricular dysfunction. Six patients were studied. Diagnosis of pulmonary embolic disease was made by ventilation perfusion scanning and/or CT pulmonary angiography. All patients were anticoagulated with oral coumarin derivatives and none were considered suitable for pulmonary thromboendarterectomy. Pulmonary hypertension was diagnosed by right heart catheterisation and each patient had Medical Research Council (MRC) dyspnoea score and New York Heart Association (NYHA) class noted and 2D echocardiography prior to commencement of sildenafil 50 mg three times a day. After 6 weeks of sildenafil therapy, right heart catheterisation and 2D echocardiography were repeated, and MRC dyspnoea score, NYHA class and exercise capacity were recorded. All patients demonstrated an improvement in mean pulmonary artery pressure, mean pulmonary capillary wedge pressure, MRC dyspnoea score, NYHA class and gas transfer. No adverse effects of sildenafil were noted. Our data suggests that sildenafil is an effective and well-tolerated therapy for patients with severe pulmonary hypertension associated with pulmonary thromboembolic disease and impaired left ventricular function, producing beneficial effects as early as 6 weeks.

Medium-term follow-up after deployment of ultraflex expandable metallic stents to manage endobronchial pathology.

BACKGROUND: Between March 1997 and March 2004 we deployed 80 Ultraflex metallic expandable stents (Boston Scientific, Waterson, MA) in 69 patients under direct vision using rigid bronchoscopy. We report our medium- to long-term experience in patients for whom these stents were deployed. METHODS: To date 15 patients have been followed for more than 1 year (median 41 months, range 12 to 83 months) after stent deployment. Indications for stenting in these patients were neoplasia (5), stricture (5), airway malacia (1), iatrogenic tracheal tear (1), and compression from an aortic aneurysm (1), a right interrupted aortic arch (1), and a right brachiocephalic artery aneurysm with tracheomalacia (1). Ten tracheal stents (9 covered, 1 uncovered) and 10 bronchial stents (8 uncovered, 2 covered) were inserted, and 5 patients received two stents. RESULTS: Five of these patients experienced no long-term problems. Complications included troublesome halitosis (5), which was difficult to treat despite various antibiotic regimes; granulation tissue formation above and below the stent that was successfully treated with low-power Nd:YAG laser therapy (7); and metal fatigue (1). We did not encounter stent migration. CONCLUSIONS: We conclude that Ultraflex expandable metallic stents have an important role in the management of selected patients with diverse endobronchial pathologies and are well tolerated in the long-term. Although associated granulation tissue can be successfully treated with Nd:YAG laser, halitosis can be a difficult problem to address.

Expression and targeting of human fibroblast activation protein in a human skin/severe combined immunodeficient mouse breast cancer xenograft model.

Antigens and receptors that are highly expressed on tumor stromal cells, such as fibroblast activation protein (FAP), are attractive targets for antibody-based therapies because the supporting stroma and vessel network is essential for a solid neoplasm to grow beyond a size of 1-2 mm. The in vivo characterization of antibodies targeting human stromal or vessel antigens is hindered by the lack of an appropriate mouse model system because xenografts in standard mouse models express stromal and vessels elements of murine origin. This limitation may be overcome by the development of a human skin/mouse chimeric model, which is established by transplanting human foreskin on to the lateral flank of severe combined immunodeficient mice. The subsequent inoculation of breast carcinoma MCF-7 cells within the dermis of the transplanted human skin resulted in the production of xenografts expressing stromal and vessel elements of human origin. Widespread expression of human FAP-positive reactive stromal fibroblasts within xenografts was seen up to 2 months posttransplantation and postinjection of cells. Human blood vessel antigen expression also persisted at 2 months posttransplantation and postinjection of cells with murine vessels coexisting with the human vascular supply. The model was subsequently used to evaluate the biodistribution properties of an iodine-131-labeled humanized anti-FAP monoclonal antibody (BIBH-7). The results showed high specific targeting of the stromal compartment of the xenograft, indicating that the model provides a useful and novel approach for the in vivo assessment of the immunotherapeutic potential of molecules targeting human stroma and angiogenic systems.

Fibroblast activation protein: differential expression and serine protease activity in reactive stromal fibroblasts of melanocytic skin tumors.

Growth and metastasis of solid neoplasms require the recruitment of a supporting tumor stroma. A highly consistent trait of tumor stromal fibroblasts in most epithelial cancers is the induction of fibroblast activation protein (FAP), a member of the serine protease family. Recently it was demonstrated that FAP has both dipeptidyl peptidase and collagenolytic activity capable of degrading gelatin and type I collagen. In this study, we describe the expression and enzyme activity of FAP in benign and malignant melanocytic skin tumors. FAP-positive fibroblasts were detected immunohistochemically in the reactive stroma of all melanocytic nevi tested. In primary and metastatic melanomas an upregulation of FAP expression in the reactive mesenchyme could be observed. Whereas 30% of the nevi revealed additional FAP expression on subsets of melanocytic cells, melanoma cells from primary and metastatic melanomas were FAP negative. This may indicate a possible role for FAP in the control of tumor cell growth and proliferation during melanoma carcinogenesis. Consistent with this in vivo expression pattern FAP enzyme activity could be detected by a specific immunocapture assay in extracts of melanocytic nevi and melanoma metastases, whereas no significant activity was detectable in normal adult skin. Strong protein expression of FAP was observed in patterned structures restricted to a subset of the melanoma metastases. Our findings that these FAP-positive structures showed no overlap with endothelial cell surface markers, nor with various melanoma antigens, suggest that FAP is a marker for specific stromal-cell-derived patterns in cutaneous melanoma metastases.